Anti-inflammatory effect of combining fish oil and evening primrose oil supplementation on breast cancer patients undergoing chemotherapy: a randomized placebo-controlled trial

Breast cancer is the most common malignant tumor and one of the leading causes of cancer-related death in women throughout the world. This study is a parallel, randomized, double-blind, controlled, 12-week supplementation trial, investigating the anti-inflammatory effects of dietary intake of fish oil and evening primrose oil (EPO), in patients with breast cancer undergoing chemotherapy. The primary outcomes were changes in the nutritional status and inflammatory cytokines of patients during the study. The secondary outcomes were changes in hematological and biochemical parameters and fatty acid profile. Of the 32 eligible patients, half of them is randomly assigned to a treatment arm with fish oil and EPO (n = 16), or a control arm (n = 16) with mineral oil as a placebo. The intervention group was taking 2 gel capsules of fish oil and 3 gel capsules of EPO (400 mg eicosapentaenoic acid, 600 mg docosahexaenoic acid, and 351 mg gamma-linolenic acid) fish oil and evening primrose oil for 12 weeks, during their chemotherapy. The control/placebo group was taking 5 gel capsules of 1g of mineral oil. One of the patients dropped out due to discontinuation of the treatment (in the placebo group) and two did not show up at the post-treatment measurements (in the intervention group), thus, 29 women completed the study. The results showed an increase in plasma levels of docosapentaenoic acid (22:5n-3), docosahexaenoic acid (22:6n-3), total n-3PUFA, vaccenic acid (18:1n-7), and a decrease in n-6/n-3 PUFA ratio in the intervention group. An increase in the plasma level of dihomo-gamma-linolenic acid (20:3n-6) was observed in the placebo group. There was no difference in plasma levels of interleukin (IL) IL-8, IL-10, and tumor necrosis factor-alpha, while the level of IL-6 decreased in both groups and was significantly lower in the intervention group at the end of the study. In conclusion, this supplementation improved the PUFA status and decreased the level of IL-6 in breast cancer patients undergoing chemotherapy. Consequently, this treatment may help reduce cancer complications resulting from impaired lipid metabolism and inflammation. ClinicalTrials.gov Identifier: NCT03516253. Date of registration 04/05/2018.


Biochemical and hematological parameters in intervention and placebo groups.
presents values of hematological parameters, at baseline and after 12 weeks. The count of leukocytes and erythrocytes, and the level of hemoglobin significantly decreased throughout the intervention period in both groups, without inter-group variabilities. Regarding biochemical parameters, all remained stable in both groups during the study (Supplementary Table S1).
Fatty acid profile and estimated activity of desaturase and elongase in intervention and placebo groups. The proportions of fatty acid changed in both groups. As expected, the supplementation with fish oil and EPO led to significant changes in FA profiles in the intervention group, resulting in increased plasma levels of n-3 PUFA (docosapentaenoic acid (22:5n-3), DHA, and total n-3PUFA), followed by a decreased palmitoleic and oleic acid as well as the n-6/n-3PUFA ratio (p < 0.01) (Table 4). Interestingly, no differences in GLA concentration were found, but the concentration of docosatetraenoic (DTA, 22:4n-6) acid, as the last product of GLA metabolism, was increased after the supplementation period. Some changes were also detected in the placebo group. Namely, the level of DGLA (20:3n-6) was significantly higher at the end of the study (Table 4).
Comparisons between groups at the end of the study, adjusted for baseline values, showed significantly higher levels, of DPA, DHA, n-3 PUFA, and total PUFA, in the intervention group. Additionally, lower levels of palmitoleic and oleic acids, DGLA, as well as an n-6/n-3 PUFA ratio (Table 4), as well as the lower estimated activity of SCD-16 were also noted in the intervention group after 12 weeks of treatment (Supplementary Table S2).  (Table 5). Additionally, the intergroup comparisons at the end of the study adjusted for baseline levels revealed a significantly (p < 0.01) lower concentration of IL-6 in the intervention group compared to the control group.

Discussion
This randomized double-blind controlled study investigated the effects of supplementation with an anti-inflammatory combination of n-3 and n-6 PUFA vs. placebo on body composition, lipid metabolism, and inflammatory response in breast cancer patients receiving the adjuvant chemotherapy during the period of 12 weeks. Although chemotherapy commonly leads to changes in weight, BMI, waist circumference, and body fat percentage 19,20 , in this study there was no change in these parameters during the first 12 weeks of adjuvant chemotherapy in either of the groups. Freedman et al. have shown that although some women with BRC did not experience significant changes in weight or BMI after chemotherapy, they had unfavorable changes in body composition (decreased fat free mass and increased percentage of body fat) 21 . Although gains in weight and/or waist circumference are positively associated with baseline values of these parameters 22 , premenopausal women display a significantly higher risk for body fat percentage gains compared to postmenopausal women 20 , during chemotherapy. Maintenance of all anthropometric parameters and body composition in all participants of our study could probably be explained by the fact that only post-menopausal women were included. Moreover, body weight changes also depend on other variables such as the type and length of chemotherapy 23 , decreased physical fitness, anxiety, depression and appetite change 24 . Further, the literature data have shown the most prominent positive effects of supplements in cancer patients with cachexia 25 . However, all the patients included in this study had BRC at early stages, and the effects of supplements are not significant, but the follow-up of these patients could show if the nutritional intervention at early stages prevents nutritional decline in the future. This is important since it will show if the supplementation at early stages should be included in the BRC therapy.
Since leukopenia and neutropenia are common side effects of many cytotoxic agents 26 , often leading to delayed cycles of chemotherapy in cancer patients, we also followed changes in hematological parameters during this study. Although n-3 PUFA has a proliferative effect on lymphocytes 27 , and improves white and red blood cell counts in mice under chemotherapeutics 28 , only a few human studies showed a beneficial effect of PUFA supplementation on hematological parameters during chemotherapy 29 . In our study, fish oil and EPO supplementation did not prevent the reduction in hematological parameters during chemotherapy in BRC patients grade IIa-IIIa, which is in line with the results of other studies 29 .  www.nature.com/scientificreports/ Biochemical parameters were stable during the 12 weeks of the study in both groups. Although some other studies have shown unfavorable change in lipid profile during chemotherapy 7 , this was not a case in the present study.
The n-3 PUFA and GLA supplementation during chemotherapy in our study participants significantly increased DPA, DHA, total n-3 PUFA, and DTA levels, and decreased the n-6/n-3 PUFA ratio in the intervention group. The optimal n-6/n-3 PUFA ratio is important not only for reducing the BRC risk 30 but also for decreasing the invasive potential of tumor cells 31,32 . Moreover, DHA can positively influence the effectiveness of chemo-and radiotherapy in BRC patients 33 . More precisely, DHA from food or supplements is quickly incorporated into all phospholipids, including those of tumor tissues, thus making the tissue more flexible and permeable to antitumor agents. Importantly, these supplements do not increase the susceptibility of healthy tissues to the drugs, e.g., to exposed toxicity of chemotherapeutics 33,34 . Therefore, the observed increase in DHA probably reflected the increase in DHA in the tumor tissue and could increase the effectiveness of chemotherapy in patients with BRC.
In the placebo group participants who remained on the standard dietary intake, only the DGLA concentration significantly increased after 12 weeks of the study, while it remained the same in the intervention group. DGLA is a precursor of arachidonic acid (AA), but also anti-inflammatory PGE1 and thromboxane A1, and could inhibit the synthesis of pro-inflammatory leukotrienes LTB4, LTC4, and LTD4. The higher concentrations of DGLA in the placebo group may indicate reduced conversion to AA and subsequent anti-inflammatory molecules. As the estimated activity of ∆5 desaturase, which catalyzes the conversion of DGLA to AA, did not change significantly during the study in both groups, the increased level of DGLA may be due to the chemotherapy-reduced conversion to anti-inflammatory molecules, further adversely affecting BRC patients and leading to inflammation.
Unlike the other members of the n-6 PUFA family, GLA exhibits anti-inflammatory, vasodilating, and anti-aggregation effects. In addition, GLA has a cytotoxic effect on tumor cells 35 , inhibits angiogenesis 36 , and stimulates apoptosis of breast, pancreatic, colon, and brain cancer cells 37,38 . In vitro studies have shown that GLA improves the effectiveness and reduces the side effects of antitumor therapy, and the response time to the therapy 39 . Despite the promising results, few interventional studies have addressed GLA supplementation in BRC. To the best of our knowledge, this is the first study that examined the effect of simultaneous supplementation www.nature.com/scientificreports/ with fish oil and EPO in women with breast cancer on adjuvant chemotherapy. In our intervention group the level of DTA (adrenic acid), as the final product of GLA conversion, was significantly increased after the supplementation, suggesting that DTA could inhibit AA release and formation of the LTB4 40 , leading to the decrease of inflammation in vivo. All the observed changes in PUFA have shifted the FA profiles in the intervention group towards anti-inflammatory profiles. It is worth to mention that although EPO contains 72% of linoleic acid (18:2 n-6), its effect in the supplemented group is not exerted, since the amount of LA in EPO is not significant in comparison to the dietary intake of this fatty acid. Another beneficial effect of the supplementation, in this study, is decreased stearoyl CoA desaturase (SCD-16) activity and reduced levels of oleic and palmitoleic acid, since the carcinogenesis is closely related to the increased activity of enzymes involved in the biosynthesis of monounsaturated fatty acid (MUFA). Namely, the overexpression of SCD, which is responsible for the conversion of saturated fatty acids into MUFA, has been confirmed in HER2 + BRC 41 and leads to increased MUFA, mostly oleic acid, in cell membranes. Oleic acid stimulates carcinogenesis by promoting the growth and migration of cells, especially those with high metastatic potential, as confirmed in HGC-27 gastric cancer and MDA-MB-231 BRC cell lines 42 .
In many inflammatory diseases, fish oil supplements are used as complementary therapy as they reduced the production of TNF-alpha, IL-1β, and IL-6 by mononuclear cells [43][44][45] . In this study, the IL-6 concentration dropped after 12 weeks of chemotherapy in both groups, but the decrease was much more prominent in the intervention group. This result demonstrates strong anti-inflammatory potential of the fish oil-EPO combination in BRC patients on chemotherapy. The changes in IL-10, IL-8, and TNF-a were not significant, possibly due to high inter-individual variabilities. However, we cannot rule out the possibility that there were modifications of these cytokines at local levels, e.g., in the tumor tissue, although they were not sufficient to modify the systemic levels. Similar findings have been reported in newly diagnosed BRC 46 . Nevertheless, this study has   Table 3. Hematological parameters of the breast cancer patients on adjuvant chemotherapy at baseline and after 12 weeks (end of the study), Mean ± SD. Leukocytes (Le), Erythrocytes (Er), Hemoglobin (Hb), Platelets (Plt). *p < 0.05, **p < 0.01, ***p < 0.001 indicates a statistically significant difference from the baseline for the same group. p values represent statistically different between the intervention and placebo groups after the treatment.

Placebo group-the end (N = 15) p
Le (× 10 9 /l) 5 www.nature.com/scientificreports/ some limitations. First, this study was conducted at a single center, affecting generalizability. Second, the study population was relatively small.
In conclusion, supplementation with fish oil and EPO of BRC patients simultaneously with chemotherapy for 12 weeks, led to significant changes in fatty acid profiles of plasma lipids and lowered levels of IL-6, suggesting a beneficial effect on lipid metabolism and inflammatory response. Additional clinical trials with a higher number of participants and long-term supplementation are required to confirm the present results.

Method
Study population. This parallel, randomized double-blind placebo-controlled study included postmenopausal women with BRC, who have started adjuvant chemotherapy by anthracycline models. Of the 60 patients screened between February 2019 and May 2021 for entry into the trial, 28 did not meet the criteria for randomization. The patients (N = 32) between 45 and 70 years of age, were recruited, from the Military Medical Academy of Belgrade, Serbia and were equally randomly assigned to be given either EPO and FO (N = 16) or mineral oil (N = 16). One of the patients in the placebo group dropped out due to discontinuation of the treatment, and two did not show up at the post-treatment measurements (in the intervention group). A total of N = 29 patients remained in the study. Eligible patients were postmenopausal women with histopathological diagnosis of breast Table 4. Plasma fatty acids profile from breast cancer patients on adjuvant chemotherapy at the start and the end of the study. The values are means ± SD. SFA-saturated fatty acid, MUFA-monounsaturated fatty acid, PUFA-polyunsaturated fatty acid. *p < 0.05, **p < 0.01, ***p < 0.001 indicates a statistically significant difference from the baseline for the same group. p values represent statistically different between intervention and placebo groups after the treatment.     Study design. This trial was a randomized 1:1 double-blind, placebo-controlled, parallel-group study conducted at the MMA and the Institute for Medical Research in Belgrade. The trial was registered at Clinical trials, NCT03516253 (Date of registration 04/05/2018). Random allocation was done by computer-generated permuted blocks of 4. The statistician who was not involved in the study generated a random allocation sequence, while the investigators enrolled participants, and assigned treatments. The patients were randomly assigned in a 1:1 to receive either supplements or a placebo. All capsules were identical in appearance, they were prepacked in bottles and consecutively numbered for each woman according to the randomization schedule. Patients and researchers were unaware of assignments to supplements and placebo groups until the statistical analysis was completed. The supplementation period started at the same time as chemotherapy. During 12 weeks of intervention, patients consumed two omega-3 Cardio capsules and three evening primrose oil capsules per day (Natural Wealth®, New York, USA), providing a total of 1000 mg EPA + DHA and 351 mg GLA. Compliance was assessed by the empty bottles that the participants returned at the end of the 12-week intervention period. Outcomes. The primary outcome was a change in nutritional status and level of interleukins in women with BRC during the study. The secondary outcomes were changes in hematological and biochemical parameters, fatty acid profile, in both groups during the study. Sample size. The sample size calculation was performed based on a study by Bougnox et al., which assessed the effect of DHA in BRC patients during chemotherapy 47 . Assuming a hypothesis that up to 5% of placebo group would have a positive response in fatty acid, inflammatory or nutritional parameters, we calculated that a minimum sample of 14 subjects in each group would allow the detection of differences caused by supplementation with n-3 PUFA, with a 80% power and 5% significance level.
Anthropometry and biochemical assays. Anthropometric measurements (weight, height, and calculated BMI) were taken at baseline and at the end of the study, via standardized methods. Blood samples for all analyses were obtained in the morning, after an overnight fast, at the beginning and at the end of the intervention. Fasting serum glucose and lipids (triglycerides (TG), total cholesterol, high-density lipoprotein (HDL)cholesterol low-density lipoprotein (LDL)-cholesterol) concentrations and hematological parameters were measured on the same day the samples were collected using clinical chemistry automated analyzer (Dimension RxL Max and ABX MICROS 60) and commercial Roche and ABX diagnostics kits, according to the manufacturer's instruction. Blood collected in the tubes with EDTA was centrifuged to separate plasma and red blood cells and plasma was stored at − 80 °C until the determination of FA profiles.
Analysis of the fatty acid profile of plasma lipids. Fatty acids from total plasma lipids were isolated as previously described 47,48 . Fatty acids were directly trans-esterified with 3 N HCl in methanol, at 85 °C for 60 min. Fatty acid methyl esters were analyzed by gas chromatograph SHIMADZU 2014 which was equipped with capillary column RESTEK Rtx 2330. The temperature program was 140-210 °C for 3°/min. Individual FA was identified by comparison with retention time of FA methyl esters commercial standards PUFA-2 (Supelco, Inc., Bellefonte, Pennsylvania, USA). Methyl esters from oils were prepared in the same way. Oils were analyzed in triplicate. The results are presented as a percentage of the total FA.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.